The technology of specific binding diagnostic reactions and reagents generally has developed rapidly in the past two decades, and continues to develop at a rapid pace. Radiommunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs), for example, have become widely known and are described in numerous tests, treatises, scientific papers and patents. ELISAs have become commonplace and achieved great importance in medicine and in scientific research since the pioneering work begun by Engvall, E., and Perlmann, P., et al, Immunochem (1971) 8:871-874; and the work of Schuurs and coworkers; see, e.g., Van Weemen, FEBS Letters (1971) 15:232-236, and several U.S. Patents naming Schuurs et al as inventors; see, e.g., U.S. Pat. Nos. Re. 31,006, 3,654,090, 3,839,153, 3,850,752, 3,862,302, 3,862,928, 3,879,262, and 4,016,043. Monoclonal antibodies in enzyme immunoassays are well known; see, e.g., the work of Herzenberg and of Engvall, and others and the later work of David et al, U.S. Pat. No. 4,376,110. DNA probes and biochemical and biological probes generally exhibit the ability to bind specifically and are of great current interest as specific binding pairs.
ENZYME IMMUNOASSAY, Ishikawa, M. D., Tadashi, Kawai, and Kiyoshi, Miyai, eds, IGAKU-SHOIN, New York 1981, describes in considerable fundamental detail the principles and practices involved in enzyme immunoassays. Reference is also made to other texts and treatises in the field, such as IMMUNOCHEMICAL METHODS IN THE BIOLOGICAL SCIENCES: ENZYMES AND PROTEINS, Mayer, R. J., and Walker, J. H., Academic Press, New York 1980; QUANTITATIVE ENZYME IMMUNOASSAY, Engvall, E., and Pesce, A. J., Blackwell Scientific Publications, London (Scandinavian Journal of Immunology, 1978); THE ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) A guide with abstracts of microplate applications, Voller, A., Bidwell, D. E., and Bartlett, A., Dynatech Laboratories, Inc., 1979; and the references cited therein for a comprehensive disclosure of the principles and usual practices involved in enzyme immunoassay.
Various approaches have been described for carrying out enzyme immunoassays. The early ELISAs were what is commonly called a "competitive" assay in which the enzyme labeled antigen or antibody competed with the antigen or antibody to be determined for a reaction site on a bead, pad or surface to which one member of an immunologically coupling pair was attached. Later, the "sandwich" assay became popular. In the sandwich assay, the antibody or antigen to be determined was "sandwiched" by an immunochemical reaction between a solid surface treated with an immunological species reactive with the species to be determined and the same or a different reactive immunological species which has been coupled to an enzyme label. The principles of these types of ELISAs are discussed by Belanger, L., Scand J Immunol, (1978) 8:Suppl. 7, 33-41; (Chapter 4 in QUANTITATIVE ENZYME IMMUNOASSAY, supra).
Many forms of solid supports to which one member of an immunochemical couple, e.g., antigen-antibody or hapten-antibody couple, have been disclosed. A common early form of solid support was a plate, tube or bead of polystyrene which was well-known from radioimmunoassay (RIA) work to bind certain immunological species. Filter paper, glass, various plastics (chemical polymers), and other solid support surfaces have been used for many years. Examples of such a system which used antibody (or antigen) coated polystyrene beads are described by Bohn et al, in U.S. Pat. No. 4,424,279, Jan. 3, 1984; and U.S. Pat. No. 4,458,020, July 3, 1984, in which the coated beads are utilized in unique configurations.
Several disclosures are directed to assays which employ passage of the sample to be tested through a solid membrane or support.
Tom et al, U.S. Pat. No. 4,366,241, disclose an apparatus for an immunoassay which includes a multiple layered construction in which the sample solution flows into an enclosure through an immunoabsorbing disk which has antibody or antigen bound to it. The solution flows from the disk through a membrane spacer which is in contact with the disk and into a bibulous strip of cellulose or paper which extends through the enclosure to a level above the sample into which the apparatus is inserted during use.
U.S. Pat. No. 4,632,901, to Valkirs et al, discloses a device and method for immunoassays in which the sample flows through the thickness of a membrane to an absorbent mass. Antibody is bound to less than the total surface of the membrane and binds antigen in the antibody coated area. Conventional ELISA techniques are used to detect the sample bound to the supported antibody.
Various configurations for self-contained assay systems have also been described; for example, Deutsch, A., and Platt, H. A., U.S. Pat. No. 4,522,923, describe a device which comprises a container, at least two water-soluble barriers, subdividing the container into at least three superimposed chambers, and different biologically active substances in each chamber. Upon introduction of an aqueous biological sample to be tested into the topmost chamber, the sample successively mixes with the contents of the chambers, the contact time in each chamber being a function of the water solubility of the barriers. The system is designed to give a color reading in the final chamber. This provides a method for conducting immunochemical reactions in a self-contained sealed unit that requires only the addition of an unknown sample and water, and thus provides an assay system that is safe and accurate even when used by an individual who is not technically trained. Specifically at least one chamber contains an antigen, antibody, or an enzyme, or their conjugates. Preferably the antibody is directed against human chorionic gonadotropin hormone. Specifically the substances in the chambers represent color-change immunochemical reactions, e.g., home testing of blood or urine for pregnancy.
Barnett, B., W08606488, describes a diagnostic test kit which has a central well for receiving a sample to be analyzed. Several reservoirs holding predetermined quantities of reagents are located in a block which surrounds the sample well and are connected to it via bores. Initially the reagents are retained in the respective reservoirs by membranes but the contents of a reservoir can be discharged by rupturing the membrane. The reservoirs are formed by resilient domes which are depressed manually to rupture the membrane and serve to transfer the reagent to the sample well. The test kit is used for screening, chemical or clinical analysis of blood, urine, swimming pool water, drinking water or soil. The test kit reduces the chance of human error in the sequential addition of reagents to a sample.
Graham, H. A., Olekna, D. J., Hawk, J. B., and Kebles, D. B., EP0022669, describe a test in which red blood cells are rapidly tested for the presence of antigens O, C, c, E, e or K by mixing them with an antibody reagent (A) and, without incubation, examining them for agglutination. (A) comprises reduced S-alkylated IgG antibody against the appropriate antigen which at least meets FDA standards for potency and specificity.
Deutsch, M. E., and Mead, L. W., U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537, describe a test strip for determining a characteristic of a sample comprises a length of material capable of transporting a developing liquid by capillarity and having zones between its ends for receiving the sample and holding reagents. The strip is used for performing binding assays, particularly those in which a radioisotope is used as a label, such as radioimmunoassays. Minute sample sizes may be used. The strip is capable of application to analytical methods having sensitivities below 0.1 mg/ml.
Friedenberg, R. M., FR 2537724, describes a dry indicator apparatus for drugs-of-abuse testing of physiological liquid test solutions. A non-bibulous capillary flow membrane vehicle matrix is impregnated with dry chemical colorants. When these are placed in contact with the test solution the colored reagent indicates the type of drug present even in low concentrations. The test is a qualitative and quantitative indicator for the presence of abused drugs, such as barbiturates, amphetamines, methadone, morphine, cocaine, codeine, dilaulid and tranquilizers of the diazepam type. The physiological fluids tested include urine, whole blood, plasma, sweat and tears.
Lipp, V., and Buck, R. L., EP 0206779, describe an apparatus for detecting antinuclear antibody in a biological sample comprising a solid support having adhered nuclei isolated from eucaryotic cells. Preferably underlying the nuclei on the solid support is a coating, e.g., of nuclear antigens, which is unreactive with antibodies to non-nuclear antigens and which, like nuclei serves to bind antinuclear antibodies in the sample. The apparatus permits the screening of human serum for the presence of antinuclear antibodies in a system featuring speed, simplicity, sensitivity and capacity for automation. Medical disorders characterized by the presence of antinuclear antibodies include systemic lupus erythematosus, mixed connective tissue disease, Sjogren's syndrome and scleroderma.
Deutsch, A., Sheets, E. J., and Rhodes, J., EP 0189925, describe a kit which comprises (a) a vessel, (b) a capillary-active wick extending from the interior of the vessel so as to wick a liquid out of the interior of the vessel, a portion of the wick carrying an immobilized immunological component selected from (i) antigen and (ii) antibody, (c) a first reagent comprising an enzyme conjugated to an immunological component selected from (i) antibody and (ii) antigen specific to (i) or (ii) respectively of (b) and (d) a substrate for the enzyme. In place of the enzyme and substrate a fluorescent label may be used. Antigens which can be tested for include dilantin, testosterone and progesterone. If the sample contains the antigen, it will combine with the antibody-enzyme while moving along the wick so that when this mixture subsequently wicks through the antigen-wick, there will be no free antibody-enzyme to bind to the antigen on the wick so it will pass out of the wick.
Friedenberg, WPO Int. Pub. No. WO 84/02397, also describes an immunoassay in which the reactions occur in the liquid phase as the sample moves through a paper support, the rate of movement being one parameter used in identifying constituents.
Campbell, U.S. Pat. No. 3,893,808, describes a strip of filter paper treated in bands with a chemical reagent, iodine, into which a sample of gasoline suspected of containing lead is wicked from one end and a developing reagent, dithizone, is wicked into the pretreated bands.
Alberty et al, U.S. Pat. No. 3,895,914, describe another chemically treated test strip in which chemical reagents are applied in bands or zones on a strip for detecting barbituric acid.
While the prior art teaches the use of wicking bibulous materials as carriers for specific binding reagents, these apparatus and methods rely principally upon the ability of the carrier to imbibe the liquid and often to enter into the reaction. The use of bibulous materials is of great value in some methods, but presents serious limitations as well, in reduced sensitivity and in the nature of the reagents and analytes which may be used or determined. The present invention utilizes a non-bibulous material in which the liquid flow is isotropic and flows laterally in the material by capillary action, thus presenting a system in which the solid membrane provides a vessel for the liquid but does not imbibe or otherwise enter into or interfere with the specific binding reactions.